J Parasitol: Vol. 14, No. 1, Jan-Mar 2019, pp.167-173

Iran J Parasitol

Tehran University of Medical Open access Journal at Iranian Society of Parasitology Sciences Public a tion http:// ijpa.tums.ac.ir http://isp.tums.ac.ir http://tums.ac.ir

Short Communication

Prevalence and Risk Factors of Toxoplasma gondii Infection among Pregnant Women in , South of Iran

Seyedeh Zahra KHADEMI 1,2, *Fatemeh GHAFFARIFAR 1, Abdolhosein DALIMI 1, Parivash DAVOODIAN 3, Amir ABDOLI 4,5

1. Department of Parasitology, Faculty of Medical Sciences, Tarbiat Modares University, Tehran, Iran 2. Department of Biology, Payam Noor University, Tehran, Iran 3. Infectious and Tropical Disease Research Center, Hormozgan Health Institute, Hormozgan University of Medical Sciences, Ban- dar Abbas, Iran 4. Department of Parasitology and Mycology, School of Medicine, Jahrom University of Medical Sciences, Jahrom, Iran 5. Zoonosis Research Center, Jahrom University of Medical Sciences, Jahrom, Iran

Received 19 Oct 2017 Abstract Accepted 11 Jan 2018 Background: Toxoplasmosis can cause miscarriage or complications in the fetus. Diagnosis and treatment of this disease by anti-parasitic drugs especially in early pregnancy can help to prevent fetal infection and its complications. This study

Keywords: aimed to determine T. gondii infection in pregnant women, evaluate risk factors in Toxoplasma gondii; the transmission of the disease and congenital toxoplasmosis. Pregnant women; Methods: Overall, 360 sera of pregnant women from 5 cities in the Hormozgan Prevalence; Province in southern Iran with different climate were evaluated from 2015-2016 Iran for T. gondii infection by using ELISA method and positive cases of IgM and IgG were tested again using Avidity IgG ELISA. All cases were evaluated according to climate, acute and chronic of toxoplasmosis, number of pregnancy and abortion,

epidemiological factors and food habits. *Correspondence Results: Among 360 specimens T. gondii IgG + IgM antibodies were found posi- Email: tive in 0. 8% subjects and also 27% of samples had IgG seropositivity. A significant [email protected] relationship was observed between age, sampling place, consumption of raw and half cooked meat, history of contact with cats, abortion history, number of chil- dren, and parity with IgG positive. In Avidity IgG ELISA test, 13 people with low avidity, 3 people with borderline avidity were reported. Conclusion: 72. 2% of the population had no antibody against the disease that this could be a warning to the people and requires education of preventive and prenatal care and routine screening of women at childbearing age.

167 Available at: http://ijpa.tums.ac.ir Khademi et al.: Prevalence and Risk Factors of Toxoplasma gondii Infection …

Introduction

ongenital toxoplasmosis is an im- is particularly useful in detecting new cases as portant infectious cause of abortion a complementary approach. The primary or C and pregnancy complications world- immature antibodies have low affinity, but wide (1). Toxoplasmosis is influenced by some affinity increases with disease proceed and will factors such as environmental conditions, nutri- stay for weeks and months. Avidity index de- tional and cultural habits, and hygiene. Climate pends on the duration of infection (7). condition can play vital role in oocysts survival. According to a meta-analysis, the overall se- The prevalence of T. gondii infection in hot and roprevalence rates of T. gondii infections is 39. humid climates is higher than warm and dry 9% (95% CI: 26. 1-53. 7) among childbearing areas and is also low in Polar Regions (2). age women in Iran (9). Moreover, two previous "Vertical transmission of T. gondii to the fe- studies in (capital of Hormozgan tus occurs predominantly in women who ac- province) showed seroprevalence rates of 49% quire the infection at the first time during (10) and 34% (11) in pregnant women. Since pregnancy "(3). Congenital toxoplasmosis can there is no comprehensive study on the preva- lead to a wide variety of manifestations ac- lence of toxoplasmosis in different cities of cording to gestational age of mothers, while, Hormozgan Province, the present study inves- severe clinical signs are more common in tigates the prevalence of T. gondii infection and women whose infection was acquired during related risk factors in a number of pregnant early gestation (4). In contrast, the risk of ver- women referring to health centers in Hor- tical transmission is increase with the gesta- mozgan Province in southern of Iran. Addi- tional age, so the highest rates of transmission tionally, IgG avidity test was used as occur in the last gestation. At this stage, the complementary test in the women who were infection is usually asymptomatic but may de- positive for IgG and IgM antibodies to T. gondii velop clinical such as neurological disorders and chorioretinitis at a later age (5). The annu- Materials and Methods al incidence of congenital toxoplasmosis was estimated in the world at 190100 cases. Metic- Area of study ulous information on the ''prevalence and risk Hormozgan Province is located in the north factors of infection" with T. gondii are required of the Hormuz Strait in southern Iran and to plan proper "prevention measures" against cover an area of about 70,697 square kilome- infection during pregnancy and congenital ters. This province has a very hot and humid transmission (6). climate (ranging between 30-49 °C and hu- A number of serological methods have been midity of 90%-100% in summers), with an developed to diagnose T. gondii infections (7). average annual rainfall of 180 mm (12,13). However, screening of IgG and IgM antibod- ies by ELISA method is a routine diagnostic Sample size method in clinical laboratories. IgM antibodies The study population was pregnant women appear in the first week of infection and reach who referred to the health centers in Hor- at the maximum level after 3 wk of infection. mozgan Province from 2015-2016. The sam- IgG antibodies usually appear two weeks after ple size was calculated by the following for- infection and reach to a peak within 8 to 10 mula and according to the previous studies in wk of infection. The presence of IgG and Bandar Abbas that reported the seropreva- absence of IgM indicates the history of lence of 38% among pregnant women (10,11). previous infection (7,8). Avidity IgG method Accordingly, 360 serum samples were collect-

Available at: http://ijpa.tums.ac.ir 168 Iran J Parasitol: Vol. 14, No. 1, Jan-Mar 2019, pp.167-173

ed from pregnant women from 5 different RAI <40%: indication of low-avidity anti- cities of the province (Bandar Abbas, Minab, bodies. Haji Abad, , Qeshm) based on geo- RAI 40%-60%: equivocal range. graphic location and climate condition. RAI >60%: indication of high-avidity anti- N=Z2 P (1- P)/d2 Z=1. 96. P=0.38. d=0.05 bodies.

Ethical aspects PCR The study protocol was approved by the The final diagnosis of toxoplasmosis in low Ethical Committee of Hormozgan University avidity cases was performed by a 529 bp gene of Medical Sciences Ethical number: (5-HEC- which replicates 200-300 in the T. gondii ge- 94-3020). All participants were informed nome (14). about the study, and sampling was conducted with informed consent. Statistical analysis All data were analyzed by SPSS (ver. 20 Chi- Sample collection cago, IL, USA) using Chi-square, Cross tab Two milliliters of blood from women re- sand Correlate Pearson test. ferred to health centers for routine pregnancy tests were collected, and their sera were stored Results at -20 °C until test. Moreover, information about pregnant women and the risk factors of Serum samples were collected from 360 the infection were gathered by questionnaire pregnant women with a mean age of 27 yr (14- at the time of sampling. Questions focused on 55) who were at different months of pregnan- possible risk factors for infection, including cy. Table 1 shows serological findings of the presence or ownership of animals, eating pregnant women taken from different cities of habits, soil contact, and etc. Hormozgan province. The percentage of T. gondii IgG and IgM+IgG positive antibodies Serological Evaluation were 27. 8% (100/360) and 0. 83% (3/360) Conventional ELISA respectively. Three women were seropositive The presence of anti-Toxoplasma IgM and for both IgG and IgM antibodies (Table 1). IgG antibodies were screened using ELISA Table 2 shows history of abortion according assay, with an ELISA kit (Pishtaz Teb, Tehran, to antibody levels. Accordingly, total IgG se- Iran) according to the manufacturer's protocol. ropositivity was significantly increased in The positive cut-off value of IgG and IgM pregnant women with a history of abortion antibodies was defined as the upper limit of (P=0.01). Risk factors of T. gondii seropositivity the 10 and 1. 1 U/mL, respectively. describe in Table 3. Significant associations were observed between IgG seropositivity and Avidity ELISA number of pregnancies (P=0. 001), age To double check, the results an IgG Avidity (P=0.004), contact to cat (P=0.03), consump- test was conducted on 100 samples with the tion of raw or half-cooked meats (P=0.05) and IgM and IgG anti-Toxoplasma antibodies using raw vegetables consumption (P=0.03) (Table 3). Toxo IgG avidity kit (EUROIMMUN, Ger- many), according to the manufacturer's proto- PCR analysis col. The diagnostic value was defined as rela- T. gondii DNA was detected in all 13 cases re- tive avidity index (RAI). The avidity index was ported low avidity in avidity ELISA method determined as the following criteria: by using 529 base pairs gene.

169 Available at: http://ijpa.tums.ac.ir Khademi et al.: Prevalence and Risk Factors of Toxoplasma gondii Infection …

Table 1: T. gondii serological findings in pregnant women in 5 cities of Hormozgan province, southern Iran

City Bandar Abbas Minab Haji Abad Bastak Qeshm Total (%) (N=190) (%) (N=40) (%) (N=40) (%) (N=40) (%) (N=50) (%) IgG positive 46(24. 2) 10(25) 6(15) 13(32. 5) 25(50) 100(27. 8) IgM Positive 0(0) 1(2. 5) 1(2. 5) 1(2. 5) 0(0) 3(0. 36)

Table 2: Risk factors of T. gondii seropositivity among pregnant women in 5 cities in Hormozgan province, southern Iran

Risk factors Variables N (%) IgG positive N P-value (%) Age(yr) Missing value† 24(6. 7) 11(42. 3) 0. 004 <20 46(12. 8) 10(21. 7) 20-39 281(78) 72(25. 6) >40 9(2. 5) 7(77. 7) Job Missing value 22(6. 1) 11(50) 0. 626 Practitioner 31(8. 6) 7(22. 5) Housekeeper 307(85. 3) 82(26. 7) Contact to cat Missing value 22(6. 1) 11(50) 0. 03 Yes 26(7. 2) 11(42. 3) No 312(86. 7) 78(25) Raw/half- Missing value 26(7. 2) 12(46) 0. 05 meat Yes 289(80. 3) 74(25. 6) No 45(12. 5) 14(31) Raw vegetables** Missing value 25(7) 12(48) 0. 03 Yes 12(3. 3) 0(0) No 323(89. 7) 88(27. 2) Raw eggs consumption Missing value 23(6. 4) 11(50) 0. 06 Yes 95(26. 4) 23(24. 2) No 242(67. 2) 67(27. 7) *did not answer. **Washing with detergent or not. In avidity IgG method, of 100 seropositive samples, 13 had low avidity

Table 3: Comparison of results of IgM ELISA with IgG avidity test in 100 serum samples taken from pregnant women in Hormozgan province

IgG Avidity IgM ELISA Total N (%) Positive (%) Negative (%) Low 3(3) 10(10) 13(13) Borderline 0(0) 3(3) 3(3) High 0(0) 84(84) 84(84) Total 3 97 100 P-value=0. 6.

Discussion to fetus is associated with a variety of symp- toms and complications (3). Toxoplasmosis is an important infection in In this study, T. gondii infection rate was 27. pregnancy that depending on the time of in- 8%. (27% IgG and 0. 83% IgM and IgG). The fection, virulence of the organism type, and highest rate (50%) of prevalence was observed number of transmitted parasites from mother in with hot and humid weather

Available at: http://ijpa.tums.ac.ir 170 Iran J Parasitol: Vol. 14, No. 1, Jan-Mar 2019, pp.167-173

and the lowest prevalence in Haji Abad with livestock of this province. Similar to study (11), hot and dry weather. A significant difference a significant relationship was found between in the infection prevalence was observed in the history of contact with cats and the pres- different climates. Probably high temperature ence of anti-Toxoplasma antibodies in pregnant weather along with higher humidity causes the women that indicates another way of trans- sporulated oocyst survival to increase. This mission in Hormozgan province. In this study, increases the probability of the infection prev- no significant association was observed be- alence among intermediate and final hosts. In tween toxoplasmosis with jobs and education. the former studies, scholars reported the simi- The combination of two sensitive methods lar results for the prevalence, indicating the inclusive IgM detection and IgG avidity highest prevalence in the regions with a higher method for T. gondii is the best way to temperate and humidity and the lowest in the determine the time of infection (25). This hot and arid districts (15). In El Salvador (16), combination can distinguish between acute with hot and humid weather, reported that up and chronic infection (26). to 90% of people over 40 yr had anti- Therefore, all three positive IgM cases had Toxoplasma antibodies. The results of other low avidity in avidity IgG method. Moreover, surveys show a prevalence of 50% to 80% in in this study, 10 subjects were negative for different areas of Brazil (17), 15% in China IgM but shown low avidity in avidity IgG (18), and 22% in London (19). method. In the follow up for these cases the In Bandar Abbas (provincial capital) the IgM were positive and the parasite DNA in prevalence was 24. 2%. In two similar studies blood was detectable. Conventional ELISA on pregnant women referred to Shariati Hos- test cannot often show the new cases of infec- pital in Bandar Abbas (11) 34.2% of women tion corrected by avidity test. A study on the were found with IgG positive and 7.9% of methods of ELISA IgG/IgM and IgG avidity IgM positive and an IgG positive rate of 41. ELISA revealed that IgG avidity is an ac- 93%, and IgM positive of zero was reported ceptable approach to distinguish between (10). The reason for the different result of this acute and chronic toxoplasmosis (27). It is a study compared to the two other studies for reliable way to screen for the disease, too, es- Bandar Abbas can come from the population pecially in the first three months of pregnancy. since they selected their samples from the The high avidity in diagnosis of chronic toxo- pregnant women referred to Shariati Hospital plasmosis is a valuable index (28). Presence of whereas in the present study in addition to T. gondii-specific IgM antibody in chronic Shariati hospital four other health centers phase of the disease sometimes leads to a were used to select the sample. wrong diagnosis and may result in an unneces- A significant relationship was found be- sary abortion. A serum sample with high avidi- tween the prevalence of T. gondii infection and ty eliminates the risk of T. gondii infection in age confirmed by studies (20) in Turkey and in the first three months of pregnancy (29). The northern Tehran (21, 22). The findings low or borderline avidity antibodies depending showed a meaningful relationship between on the immune system and the development the consumption of raw or half-cooked meats of IgG antibodies may last for months, so ap- and infection, too. Thus, probably this is one plying the results of avidity test alone can be of the ways of infection transmission in the misleading (30). province since the most amount of meat is Since no correlation was observed between provided by neighboring provinces such as the results of avidity IgG test and levels of Fars Province with 37. 5% (23) and Kerman IgG and IgM antibodies, both tests should be with 24.7% of sheep infection (24). However, used for better detection. The presence of so far, no study has been conducted on the high avidity antibodies indicates a definite

171 Available at: http://ijpa.tums.ac.ir Khademi et al.: Prevalence and Risk Factors of Toxoplasma gondii Infection …

chronic infection and suggests infection in at 5. Abdoli A, Dalimi A, Arbabi M, Ghaffarifar F. least 3-5 months. Therefore, high avidity anti- Neuropsychiatric manifestations of latent bodies are valuable in the first three to four toxoplasmosis on mothers and their offspring. months of pregnancy, but in the following J Matern Fetal Neonatal Med. 2014; months, there is no diagnostic value (29). Low 27(13):1368-74. 6. Torgerson PR, Mastroiacovo P. The global avidity indicates an acute infection, it cannot burden of congenital toxoplasmosis: a be ascertained at a time interval between acute systematic review. Bull World Health Organ. and chronic infection that there is a risk of 2013;91(7):501-8. infection for the fetus. Therefore, this doubt 7. Villard O, Cimon B, L’Ollivier C et al. can be resolved by molecular methods. Serological diagnosis of Toxoplasma gondii infection: recommendations from the French Conclusion National Reference Center for toxoplasmosis. Diagn Microbiol Infect Dis. 2016;84(1):22-33. In general, screening tests for toxoplasmosis 8. Zhang K, Lin G, Han Y, Li J. Serological diagnosis of toxoplasmosis and standardization. should be performed for pregnant women and Clin Chim Acta. 2016; 461: 83-89. for greater certainty, deterministic tests such 9. Borna S, Shariat M, Fallahi M, Janani L. as avidity IgG are proposed. Prevalence of immunity to toxoplasmosis among Iranian childbearing age women: Acknowledgements Systematic review and meta-analysis. Iran J Reprod Med. 2013; 11(11): 861-868. This study was financially supported by 10. Makiani MJ, Davoodian P, Golsha R et al. Tarbiat Modares University. The authors Seroepidemiology and risk factors of would like to thank Dr Sadraei and Dr Samiei toxoplasmosis in the first trimester among pregnant women. Int Elec J Med. 2012; 1: 12- for their kind assistance. 17. 11. Setoodeh Jahromi A, Safa O, Zare S, Conflict of interest Davoodian P, Farshid Far G. Prevalence of Toxoplasma antibodies in pregnant women The authors declare that there is no conflict referred to Shariati Hospital, Bandar Abbas, of interests. Iran, 1999-2000. J Hormozgan Uni Med Sci. 2003; 4: 25-30. References 12. Hormozgan Province. https://en.wikipedia.org/wiki/Hormozgan_Pr ovince 1. Mcleod R, Lykins J, Noble AG et al. 13. Hormozgan Province. Management of congenital toxoplasmosis. Curr http://www.hormozganmet.ir/fa/ Pediata Rep. 2014;2(3):166-94. 14. Homan W, Vercammen M, De Braekeleer J, 2. Meerburg BG, Kijlstra A. Changing climate Verschueren H. Identification of a 200-to 300- changing pathoens :Toxoplasma gondii in North fold repetitive 529 bp DNA fragment in Western Europe. Parasitol Res. 2009;105(1):17- Toxoplasma gondii, and its use for diagnostic and 24. quantitative PCR. Int J Parasitol 2000; 3. Goldsten EJ, Montoya JG, Remington JS. 30(1):69–75. Management of Toxoplasma gondii infection 15. Mostafavi SN, Jalali L. Toxoplasmosis during pregnancy. Clin Infect Dis. epidemiology in Iran: a systematic review. J 2008;47(4):554-66. Isfahan Med School. 2012;30(176): 1-15.

4. Li XL,Wei HX, Zhang H, Peng HJ, Lindsay 16. McCabe RE, Remington JS. Toxoplasma gondii. DS. A meta analysis on risks of adverse In: Editor(s): John E. Bennett, Raphael Dolin, pregnanct outcomes in Toxoplasma gondii Martin J. Blaser, Mandell, Douglas, and infection. Plos One. 2014;9(5):e97775. Bennett's. Principles and Practice of Infectious

Available at: http://ijpa.tums.ac.ir 172 Iran J Parasitol: Vol. 14, No. 1, Jan-Mar 2019, pp.167-173

Diseases. ISBN 9781455748013, 1990:2090- 24. Bahrieni M, M Fasihi Harandi, M Beigzadeh et 103. al. Risk factors analysis associated with 17. Dubey JP, Lago EG, Gennari SM, Su C, Jones seropositivity to Toxoplasma gondii in sheep and JL. Toxoplasmosis in humans and animals in goats in Southeastern Iran using modified Brazil: high prevalence, high burden of disease, agglutination test (MAT). Iran J Parasitol. 2008; and epidemiology. Parasitology. 3(1): 38-43. 2012;139(11):1375-424. 25. Petersen E , Borobio M. V, Guy E, Liesenfeld 18. Zhou P, Chen Z, Li HL, Zheng H, He S, Lin O, Meroni V, NaessensA,Thulliez P. European RQ, Zhu XQ. Toxoplasma gondii infection in multicenter study of the LIAISON automated humans in China. Parasite Vectors. diagnostic system for determination of 2011;4(1):165. Toxoplasma gondii specific immunoglobulin G 19. Dubey JP. Toxoplasmosis. In: Editors; Collier (IgG) and IgM and the IgG avidity index. J L, A Balows, M TopleySussman. Wilson's Clin Microbiol. 2005;43(4):1570-74. Microbiology and Microbial Infections, vol. 4. 26. Iqbal J , Khalid N. Detection of acute Arnold, London, Sydney, Auckland, New York. Toxoplasma gondii infection in early pregnancy by 1998: 303-318. IgG avidity and PCR analysis. J Med Microbial. 20. Doni NY, Simsek Z, Gurses G, Zeyrek FY, 2007; 56(11), 1495-99. Demir C. Prevalence and associated risk factors 27. Tanyuksel M, Guney C, Araz E, Saracli MA, of Toxoplasma gondii in female farmworkers of Doganci L. Performance of the southeastern Turkey. J Infect Dev Ctries. 2015; immunoglobulin G avidity and enzyme 9(01): 087-93. immunoassay IgG/IgM screening tests for 21. Ghadamgahi F, Bahadoran M, Shariat- differentiation of the clinical spectrum of Bahadori E, Ahmadi-Ahvaz N, Ghadrdoost B, toxoplasmosis. J Microbiol. 2004; 42(3): 211-15. Hejazi SH. Study of serological toxoplasmosis 28. Villard O, Breit L, Cimon B et al. Comparison and risk factors associated with infection in of four commercially available avidity tests for women referred to labs of Northern Tehran, Toxoplasma gondii-specific IgG antibodies. Clin Iran. J Isfahan Med School. 2013; Vaccine Immunol. 2013; 20(2): 197-204. 31(248):1258-66. 29. Liesenfeld O, Montoya JG, Kinney S, Press C, 22. Ajami A, Sharif M, Saffar MJ, Zyaee H. Remington JS. Effect of testing for IgG avidity Serological study of toxoplasmosis in women in the diagnosis of Toxoplasma gondii infection in referred to Medical Health Laboratory before pregnant women: experience in a US reference marriage, Mazandaran, 2000. J Mazandaran laboratory. J Infec Dis. 2001; 183(8) : 1248-53. Uni Med Sci. 2006;2(2): 134-7. 30. Singh S, Pandit AJ. Incidence and prevalence 23. Asgari Q, Sarnevesht J, Kalantari M et al. of toxoplasmosis in Indian pregnant women: a Molecular survey of Toxoplasma infection in prospective study. Am J Reprod Immunol. sheep and goat from Fars province, Southern 2004; 52(4): 276-83. Iran. Trop Anim Health Pro. 2011; 43 (2): 389- 92.

173 Available at: http://ijpa.tums.ac.ir